The present invention is directed to a process for culturing a variety of higher Basidiomycetes mushrooms using defined media, to produce superior yields of biologically active nutriceuticals. The nutriceutical agents are isolated by a simple one-step process, and are formulated for use as dietary supplements to achieve normal human bodily functions in general, and to control hyperglycemia in particular. Specifically, this invention relates to a method of producing a polysaccharide glucuronoxylomannan, which has medicinal properties, and a biomass rich in the essential amino acids and vitamins, from the culture broth obtained from a submerged culture of an edible higher Basidiomycetes mushroom belonging to the genus Tremella.
Mushrooms or macrofungi with distinctive fruiting bodies of sufficient size to be seen with the naked eye, include about 10,000 species of varying degrees of edibility. Approximately 100 species have been tested for cultivation and only seven to eight have been cultivated on an industrial scale. The world production of cultivated edible mushrooms in 1994 was estimated to be about five million tons and was valued at about ten billion US dollars. The most popular species of cultivated edible mushrooms include Agaricus bisporus (J. Lge) Imbach, A. bitorquis (Quxc3xa9l.) Sacc., Lentinus edodes (Berk) Sing., Pleurotus spp., Auricularia spp., Volvariella volvacea (Fr.) Sing., Flammulina velutipes (Fr.) Sing., Tremella fuciformis Berk., Hypsizygus marmoreus (Peck) Bigel., Pholita nameko (T. Ito) S. Ito et Imai, Grifola frondosa (Dicks.: Fr.) S.F. Gray, Hericium erinaceus (Bull.: Fr.) Pers., Dictyophora indusiata (Vent.: Pers.) Fischer, Stropharia rugosoannulata Far. apud Murr., Lepista nuda (Bull.: Fr.) Cooke, Agrocybe aegerita (Brig) Sing.
The cultivation of fruiting bodies of mushrooms deals with living organisms, for example, the mushroom itself and other microorganisms which may either be harmful or beneficial. Therefore, the methods employed in mushroom cultivation require modifications depending upon the region being cultivated, substrates available, environmental conditions and species of microorganisms encountered.
The cultivation of mushrooms for fruit bodies production is a long-term process needing from one to several months for the first fruiting bodies to appear. Moreover, it was found that processes for extraction of polysaccharides from fruit bodies are not considered commercially feasible, since the physicochemical properties of the products resulting from these processes were not known or regulated, Otsuka, U.S. Pat. No. 4,051,314. Submerged culturing of polysaccharide producers allows obtaining the end product of constant composition in a short period under controlled conditions using ecologically pure culture medium of defined composition.
Several species of the genus Tremella have been used in folk medicine since ancient times. These are T. fuciformis, T. mesenterica, T. aurantia, and T. cinnabarina, and all of them are characterized as xe2x80x9cstrengthening health, resisting disease,xe2x80x9d Yang Q. et al, 1989, Mushroom Science 12: 631-643. The last three species, with yellow-gold fruit bodies, are known under the common name xe2x80x9cKinjixe2x80x9d in Oriental medicine and are considered to possess equal medicinal value, Ukai S. et al., 1995, JP 7, 238, 031 A.
Tremella mushrooms belong to the so-called jelly mushrooms, which form gelatinous fruit bodies. The jelly mushrooms are a set of species from different taxonomical groups of Phragmobasidiomycetes, which are able to survive long periods of drought by drying to a horny texture. When moisture is again available, they absorb water and become gelatinous. This characteristic of jelly mushrooms is due to the presence of specific water absorbing polysaccharides that compose 60-70% of the dry fruiting body. Unlike the xcex2-1-3-glucans polysaccharides from other medicinal mushrooms, jelly mushroom polysaccharides consist of other sugars as well as glucose, and therefore belong to the class of heteropolysaccharides. A unique feature of Tremella mushrooms is that their pharmacologically active polysaccharides make up most of the structural fruit body polysaccharides while in other medicinal mushrooms pharmacologically active polysaccharides make up only a small part of the biomass. For example, in shiitake mushrooms only 31 g of lentinan was extracted from 200 kg of fresh mushrooms, Mizuno, 1999, Int. J. Medicinal Mushrooms, 1:7-27.
The main pharmacologically active substance from Tremella is the polysaccharide glucuronoxylomannan, consisting of a linear backbone of 1,3-linked alpha-D-mannose with mainly xylose and glucuronic acid in side chains. The chemical structure of Tremella glucuronoxylomannan differs among various samples of even one species, and may be in some way connected with a type of polysaccharide-based method of identification. The general proportions of xylose:glucuronic acid:mannose are given in Tremella fuciformis as 1.0:2.77:4.9; 2:1:4 in T. aurantia, and 7:1:5 in T. mesenterica, Fraser CE et al., 1973, Can J. Biochem. 51: 219-224. Some additional saccharides can be identified in different samples of T. fuciformis, such as glucose and fucose , xylobiose and fructose.
Several species of Tremella produce glucuronoxylomannan which has been shown to have hypocholesterolemic activity. Hypercholesterolemia and dyslipoproteinemia, causing high blood pressure and diabetes, are the main risk factors determining ischemic cardiopathologies and cerebral vascular accidents. Hypercholesterolemia is defined as the increase of the blood cholesterol ratio above 2 g/l. Dyslipoproteinemia coincides with the disturbance of diverse lipoprotein levels, resulting in lipid sedimentation on the arterial walls. Tremella fuciformis fruiting bodies, taken as 5% dried powder in a hypercholesterolemic diet (1.5% cholesterol, 5% fat), decreased the serum total cholesterol concentration in rats 19% after 4wk of mushroom diet consumption. Similar significant decreases in serum low-density lipoprotein (LDL) and triacylglycerol levels were observed, Cheung, PCK, 1996, Nutrition Res. 16: 1721-1725.
The antilipemic effect of polysaccharides from both T. fuciformis fruit body and pure culture has been described earlier, Janhe S. et al., 1989, J. China Pharm. Univ., 20:344-347, and it has been proposed that Tremella polysaccharides may be useful as dietary supplement in the prevention and therapy of atherosclerosis, Ryong LH et al., 1989, Drug Dev. Res. 17: 109-117. In addition, Tremella polysaccharides may have hepatoprotective functions in cases of chronic hepatitis Xiong HZ et al., 1985, Chin. J. Antibiot. 10: 363-365.
Tremella aurantia was found to have hypoglycemic activity in normal mice and in two diabetic mouse models, streptozotocin-induced diabetes and genetic diabetes. Diabetes is defined by a fasting glycemia above 1.4 g/l evaluated on two different blood samples. Insulin-dependent diabetes (IDD) corresponds to a fault in insulin secretion; cardiovascular complications are due to the important and lasting hyperglycemia causing the persistence of proteins in the urine. Tremella aurantia fruit body polysaccharide (TAP) was found to be effective in reducing hyperglycemia following not only intraperitoneal, but also oral administration (0.5 g/l TAP). Similar effects in prevention and treatment of alloxan- or streptozotocin-induced diabetic mice were shown for T. fuciformis polysaccharides, Xue W. et al, 1989, J. China Pharm. Univ. 20: 181-183; It was proposed that the mechanism of hypoglycemic activity in normal mice was at least related the increase of insulin secretion and for the acceleration of glucose metabolism Kiho, T. et al., 1994, Yakugaku-Zasshi,114:308-315.
Acid heteroglycans from T. fuciformis was found to have cytokine-stimulating activity. Four kinds of acidic heteroglycans with molecular weights from 550 to 48 KDa were isolated from the fruit bodies. These and additional fragments of their acidic hydrolysate also induced monocytes to secrete interleukin-6 with high potency, indicating that the activity may be caused by a common structure, (1xe2x86x923)-mannan in the four heteroglycans and their fragments. So, the change of molecular weight had no obvious influence on the activity of the heteroglycans, Gao Q. et al., 1996, Carbon Res. 288:135-142.
Naturally growing or artificially cultivated fruit bodies of Tremella mushrooms have been extensively used during the last decade for development of different types of Tremella health-care nutritive or medicinal food, drink or beverage (CN Patt. 1125065; 1109300; 1102305; 1099946; 1091263; 1082362; 1072829; 1066964; 1044036; 1204474; 1178122; 1114871; JP Pat. 6153879; 6339354; 60075279).
These Tremella-by products can be classified as xe2x80x9cmushroom nutriceuticalsxe2x80x9d, which are refined/partially defined extracts from either the mycelium or the fruiting body of the mushroom, which are consumed in the form of capsules or tablets as a dietary supplement or functional foods, and which have potential therapeutic applications. Chang ST, 1999, Int. J. of Medicinal Mushrooms 1: 1-7. In this way nutriceuticals differ from xe2x80x9cnutraceuticalsxe2x80x9d which are consumed as part of the normal diet and may have been modified/enriched in some way to provide health-giving benefits. Enriched compositions of nutriceuticals may thus be prepared by selecting the appropriate strains of mushroom and/or by optimizing the culture cultivation conditions. Hence the regulatory requirements for approval of nutriceuticals for human consumption may be more stringent than those for nutraceuticals.
However, it is found that nutraceutical products from fruiting bodies of medicinal mushrooms are very diverse in quality and quantity of different nutrients, and there are heretofore, no standard protocols for guaranteeing reproducible high product quality. Chang ST et al, 1999, Int. J. of Medicinal Mushrooms 1: 139-146. So, it is generally desirable to have nutriceutical compositions that are relatively uniform as to the type and levels of nutrients present in them. Generally, it has been found in cultivating different mushroom strains, that the polysaccharides extracted from the fruit bodies and from mycelia in pure cultures are not essentially the same, although both may be pharmacologically active. A slight difference was observed in xylose:glucuronic acid:mannose proportions in Tremella fuciformis polysaccharide from fruit bodies (1.0:2.77:4.9) and those obtained from pure cultures of different haploid yeast-like budding strainsxe2x80x941:0.8-1.3:2.1-3.5, Kakuta M. et al., 1979, Agric. Biol. Chem. 43: 1659-1668. The Tremella fuciformis polysaccharide had a hypocholesterolemic effect, which is characteristic for fruit bodies polysaccharide, when tested in rats with the addition of submerged culture-derived polysaccharide to a high-cholesterol diet.
Alternatively, acidic technology has been proposed to produce Tremella proteoglycan from artificially growing mycelium on a semisolid medium after fruit bodies have been removed from this substrate (CN Pat. 1071060). A pure culture of Tremella was used for production of a fermented beverage, when Tremella strains were inoculated in a liquid medium made with potato as the main raw material. The fermented liquor was directly made into a Tremella polysaccharide beverage after filtration and also could be concentrated and dried in order to obtain crude Tremella polysaccharide powder (CN Pat. 1069866; 1057954). This high-level nutritive beverage which fully embodies the nutritive and medicinal value of Tremella is proposed for enhancing the effect on human immunity and exerting antiageing, anti-tumor, or anti-hyperlipemia effects.
A special method for increasing the growth rate of Basidiomycetes species Coriolus versicolor pure culture was developed (U.S. Pat. No. 4,159,225). It was found that when dycariotic mycelium of this species was subjected to submerged culture while undergoing a mechanical treatment such as grinding or shearing in a liquid medium, the mycelium lost clamp connection, which is an intrinsic morphological characteristic, and changed into a monocariotic mycelium, and that the thus-formed monocariotic mycelium was stable and also had a unique characteristic in its extremely high propagation rate as compared with the known dycariotic mycelium.
A unique feature of some Heterobasidiomycetes species, to which the Tremella species belongs, is that their monocariotic (haploid) strains are able to grow in the form of yeast-like budding cells, and these monocariotic strains can be obtained not by grinding dycariotic mycelium but by development of monobasidiosporous cultures from the basidiospore print. This biological phenomenon was used for the production of Tremella fuciformis food and beverage, when yeast-like cells obtained by cultivating T-9 haploid strain (FERM No. 9419) in a submerged culture were blended with other components to form a food and beverage having an inhibitory action on a rise in cholesterol (JP Pat. 1020070). Heretofore, one cell cultures of Tremella have not been used to produce compounds having a hypoglycemic activity, and in particular, one cell cultures of Tremella mesenterica have not been used for any purpose.
The present invention relates to cultivation in submerged culture containing defined nutrient medium, of a one cell culture of the edible Basidiomycetes mushrooms comprising the genus Tremella including, but not limited to Tremella mesenterica, T. fuciformis, T. aurantia, and T. encepuala. 
In a first aspect, the invention provides a method of cultivating submerged cultures of one or more Basidiomycetes mushrooms having the trait to produce one or more substances having hypoglycemic activity. The use of the nutrient media of the invention, comprising a saccharide containing glucose in the molecule, an organic or mineral source of nitrogen and a variety of salts, is especially suited to enhance the production of glucuronoxylomannan, having a hypoglycemic activity, by submerged culturing of haploid yeast-like budding cultures of an edible mushroom selected from Tremella mesenterica. 
In the second aspect, the invention provides a method to concentrate the hypoglycemic compound together with mushroom cells thus enabling the simple separation of the edible one-cell biomass and exocellular polysaccharides from the fermentation broth by alcohol precipitation, thereby requiring no further extraction, concentration, purification or complex separation procedures. The simple separation of the edible Basidiomycetes from the culture broth of the present invention is followed by drying after extraction with alcohol.
In accordance with the invention, compositions including glucuronoxylomannan, a glucose lowering compound is described, which, when orally consumed or ingested, lowers the blood levels of glucose. The preventive and/or treatment method of the invention therefore involves reduction of risk posed by elevated glucose in subjects at high risk of having diabetes.
The present invention can provide methods and compositions including nutriceutical components generally beneficial for promoting health, for example, glucuronoxylomannan, vitamins, protein rich in the essential amino acids and free amino acids.
Still other objects and advantages of the invention will in part be obvious and will in part be apparent from the specification.